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nature publishing group
Pulmonary fibrosis: pathogenesis, etiology and
regulation
MS Wilson1 and TA Wynn1
Pulmonary fibrosis and architectural remodeling of tissues can severely disrupt lung function, often with fatal
consequences. The etiology of pulmonary fibrotic diseases is varied, with an array of triggers including allergens,
chemicals, radiation and environmental particles. However, the cause of one of the most common pulmonary fibrotic
conditions, idiopathic pulmonary fibrosis (IPF), is still unclear. This review examines common mechanisms of pulmonary
wound-healing responses following lung injury, and highlights the pathogenesis of some of the most widespread
pulmonary fibrotic diseases. A three phase model of wound repair is reviewed that includes; (1) injury; (2) inflammation;
and (3) repair. In most pulmonary fibrotic conditions dysregulation at one or more of these phases has been reported.
Chronic inflammation can lead to an imbalance in the production of chemokines, cytokines, growth factors, and disrupt
cellular recruitment. These changes coupled with excessive pro-fibrotic IL-13 and/or TGF1 production can turn a wellcontrolled healing response into a pathogenic fibrotic response. Endogenous regulatory mechanisms are discussed
including novel areas of therapeutic intervention. Restoring homeostasis to these dysregulated healing responses, or
simply neutralizing the key pro-fibrotic mediators may prevent or slow the progression of pulmonary fibrosis.
INTRODUCTION
Following injury it is paramount that tissue architecture is
restored to regain normal organ function. Acute inflammatory
responses that result from infection or injury can disrupt epithelial and endothelial integrity leading to edema, the recruitment
of leukocytes and angiogenesis. The resolution of inflammation
through apoptotic and phagocytic pathways often leaves minimal damage and restores normal tissue architecture. However,
common to most fibrotic conditions is the presence of a persistent irritant, which can be known agents, such as allergens, toxic
chemicals, radiation, or other persistent irritants or unknown
factors that trigger idiopathic pulmonary fibrosis (IPF). Indeed,
a dysregulated healing response can gradually evolve into a pathogenic fibrotic response when important checkpoints are missed
and inflammation becomes unrelenting. These processes can
result in a local milieu rich in chemokines, pro-inflammatory,
angiogenic, and fibrogenic cytokines, growth factors and tissue
destructive enzymes.1–3 This mélange of dysregulated processes
can result in an increased accumulation of extracellular matrix
(ECM) components and fibrotic lesions. Concurrent inflammation, tissue destruction and tissue regeneration can present
a “perfect storm” of damage and regeneration.
A tightly regulated repair response following tissue injury
is therefore critical. A well-coordinated influx of cells replace
resident tissue cells, supply essential nutrients, and reform
the tissue during a regenerative period. In some cases, this is
followed by a period of fibroplasia, with too much extracellular
matrix deposition and connective tissue formation. These events
are often associated with vascular diseases and can give rise
to many clinical conditions such as atherosclerosis, cirrhosis,
scleroderma, asthma, and various types of pulmonary fibrosis.
The regenerative process following tissue damage, despite having common mechanisms, can lead to various organ-specific
disorders. This review will focus on pulmonary fibrotic
conditions and, if known, present common regulatory
mechanisms across diseases.
The prevalence and incidence of pulmonary fibrotic diseases
are hard to estimate, given the vast array of clinical conditions.
IPF affecting 30 in 100 0004 with 34 000 new cases annually5
and allergic asthma, affecting one in five in the United States;
(http://www.cdc.gov/nchs/fastats/asthma.htm) although not
always leading to airway remodeling and fibrosis, which are
two of the most common pulmonary fibrotic diseases. In addition, there are many other fibrotic diseases of the lung including
1Immunopathogenesis Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda 20892,
Maryland, USA. Correspondence: MS Wilson ([email protected])
Received 14 November 2008; accepted 2 December 2008; published online 7 January 2009. doi:10.1038/mi.2008.85
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cystic lung disease, scleroderma, radiation and chemotherapyinduced fibrosis, granulomatous lung disease, sarcoidosis and
environmental, and smoking-associated COPD. These fibrotic
conditions are frequently fatal, with a median survival time following diagnosis of 3–5 years in the case of IPF.6
MECHANISMS OF WOUND HEALING AND FIBROSIS
A wound-healing response is often described as having three
distinct phases—injury, inflammation and repair (Figure 1).
Although not all pulmonary fibrotic conditions follow this simple paradigm, it has been a useful model to elucidate the common and divergent mechanisms of pulmonary fibrosis.
Phase I: injury
Injury caused by autoimmune or allergic reactions, environmental particulates, infection or mechanical damage often results in
the disruption of normal tissue architecture, initiating a healing
response. Inflammation following insult, can also contribute to
cellular damage and tissue destruction. Damaged epithelial and
endothelial cells must be replaced to maintain barrier function
and integrity and prevent blood loss, respectively. Acute damage
to endothelial cells leads to the release of inflammatory mediators and initiation of an anti-fibrinolytic coagulation cascade,7
temporarily plugging the damaged vessel with a platelet and
fibrin-rich clot. Lung homogenates, epithelial cells or BAL fluid8
from IPF patients express greater levels of the platelet-differentiating factor, X-box-binding protein-1, compared with COPD
and control patients,9 suggesting that clot-forming responses
are continuously activated. In addition, thrombin (a serine protease required to convert fibrinogen into fibrin) is also readily
detected within the lung and intra-alveolar spaces of several
pulmonary fibrotic conditions,10–12 further confirming the
WOUND HEALING
INJURY
INFLAMMATION
EPITHELIAL / ENDOTHELIAL
CELL DAMAGE
PLATELET ACTIVATION
FIBRIN-RICH CLOT
FORMATION
CYTOKINE, CHEMOKINE,
GROWTH FACTOR RELEASE
(MYO)FIBROBLAST
DIFFERENTIATION, EMT AND
FIBROCYTE RECRUITMENT
ANGIOGENESIS
RADIATION
ALLERGEN
INFECTION
AUTOIMMUNITY
ENVIRONMENTAL
PARTICLES
BLM-induced pulmonary
inflammation and fibrosis
REPAIR
APOPTOSIS AND
PHAGOCYTOSIS
WOUND CONTRACTION
RE-EPITHELIALIZATION
REGENERATION
Resolution and repair
CHEMOTHERAPY
Alveolus
Epithelium
Parenchyma
Vasculature
Parenchyma
Platelets
Inflammatory cells
myofibroblasts
Figure 1 Phases of wound healing. A three-phase injury and wound-healing model describes distinct phases of a successful response. (1) Injury;
many agents can cause pulmonary injury, including environmental particles, allergens, infectious agents, chemotherapy and radiation. Disruption of
epithelial and endothelial cells initiate an anti-fibrinolytic cascade, temporarily plugging the affected tissue. (2) Inflammation; circulating inflammatory
cells and fibrocytes are recruited to the injured site through chemokine gradients, supplying fibroblast-activating cytokines and growth factors. Neovascularization provides access to damaged areas and a steady stream of inflammatory, anti-inflammatory, and phagocytic cells. (3) Fibroblasts
contract and decrease the size of the wound. Inflammatory cells and -SMA + myofibroblasts undergo apoptosis, terminating collagen deposition, and
are cleared by phagocytic cells. Epithelial and endothelial cells are replaced and tissue architecture is restored.
104
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activation of the clotting pathway. Thrombin can also directly
activate fibroblasts,13 increasing proliferation and promoting
fibroblast differentiation into collagen-producing myofibroblasts.14,15 Damage to the airway epithelium, specifically alveolar
pneumocytes16 can evoke a similar anti-fibrinolytic cascade and
lead to interstitial edema, areas of acute inflammation and separation of the epithelium from the basement membrane.17
Platelet recruitment, degranulation and clot formation rapidly
progress into a phase of vasodilation with increased permeability,18 allowing the extravasation and direct recruitment of leukocytes to the injured site. The basement membrane, which forms
the ECM underlying the epithelium and endothelium of parenchymal tissue, precludes direct access to the damaged tissue. To
disrupt this physical barrier, zinc-dependent endopeptidases,
also called matrix metalloproteinases (MMPs), cleave one or
more ECM constituents allowing extravasation of cells into, and
out of, damaged sites. Specifically, MMP-2 (gelatinase A, Type
N collagenase) and MMP-9 (Gelatinase B, Type IV collagenase)
cleave type N collagens and gelatin, two important constituents
of the basement membrane.19–21 In the majority of studies, but
not all,22 MMP-2 and MMP-9 are upregulated23–26 highlighting
the tissue destructive and regenerative processes common in
fibrotic conditions.
The precise function of MMP-2 and MMP-9 was elegantly
demonstrated in a model of allergic airway inflammation and
remodeling with MMP-2 − / − , MMP-9 − / − and MMP-2 − / − MMP9 − / − double knockout mice.27,28 In these studies, the authors
demonstrated that MMP-2, and more importantly MMP-9, were
required for successful egression and clearance of inflammatory
cells out of the inflamed tissue and into the airspaces. In the
absence of these MMPs, cells were trapped within the parenchyma of the lung and were not able to move into the airspaces,
which resulted in fatal asphyxiation.
The activities of MMPs are controlled by several mechanisms
including transcriptional regulation, proenzyme regulation, and
specific tissue inhibitors of MMPs. The balance between MMPs
and the various inhibitory mechanisms can regulate inflammation and determine the net amount of collagen deposited during
the healing response.4
Phase II: inflammation
Once access to the site of tissue damage has been achieved,
chemokine gradients recruit inflammatory cells. Neutrophils,
eosinophils,29 lymphocytes, and macrophages are observed
at sites of acute injury with cell debris and areas of necrosis
cleared by phagocytes. The influence of specific inflammatory
cells on downstream fibrosis, particularly in IPF, is controversial
(30–32 and was recently reviewed33). One school of thought stems
from the observation that anti-inflammatory agents have little efficacy in the treatment of IPF34–36 and usual interstitial
pneumonia patients. Based on these observations, many investigators have suggested that inflammation per se may not be a
contributing factor in fibrosis. However, we believe the controversy reflects our limited knowledge and insight into the
causative agent(s) and mechanisms involved in IPF. The timing
of inflammatory events may determine the role played by the
MucosalImmunology | VOLUME 2 NUMBER 2 | MARCH 2009
inflammatory process. Early inflammation that is diminished at
the later stages of disease may promote wound healing and may
contribute to fibrosis. For example the early recruitment of eosinophils, neutrophils, lymphocytes, and macrophages providing
inflammatory cytokines and chemokines can contribute to local
TGF and IL-13.37–41 However, following the initial insult and
wave of inflammatory cells, a late-stage recruitment of inflammatory cells may assist in phagocytosis, clear cell debris, and
control excessive cellular proliferation, which together may
contribute to normal healing. Thus late-stage inflammation
may in fact serve an anti-fibrotic role and could be required
for successful resolution of wound-healing responses. For
example a late-phase inflammatory profile rich in phagocytic
macrophages,42 assisting in fibroblast clearance, in addition to
IL-10-secreting regulatory T cells, suppressing local chemokine production and TGF,43 may prevent excessive fibroblast
activation. Thus, the absence of inflammation observed in IPF
patients,36 and interpretation that inflammation is not involved,
may simply be a matter of timing. Indeed, corticosteroids that
inhibit endogenous suppressive and phagocytic pathways may
even be detrimental. However, It should not be forgotten that
the mechanisms leading to pulmonary fibrosis are diverse, with
immeasurable genetic, environmental and immunological interactions regulating the entire process.
The nature of the insult or causative agent often dictates the
character of the ensuing inflammatory response. For example,
exogenous stimuli like pathogen-associated molecular patterns (PAMPs) are recognized by pathogen recognition receptors, such as toll-like receptors and NOD-like receptors, and
influence the response of innate cells to invading pathogens.44
Endogenous danger signals45 can also influence local innate cells
and orchestrate the inflammatory cascade. For immunologists,
classifying the type of immune response into Type-1 (Th1 cells,
IFN, TNF, and IgG2 antibody responses, generally considered
pro-inflammatory) Type 2 (Th2 cells, IL-4, IL-5, IL-13, and IgE,
generally considered as a wound-healing response) and type 17
(Th17 cells, recently associated with pro-inflammatory conditions) based upon the T helper cell-dominant cytokine responses,
although often oversimplifying, allows for easier discussion.
The nature of the inflammatory response dramatically influences resident tissue cells and the ensuing inflammatory cells.
Inflammatory cells themselves also propagate further inflammation through the secretion of chemokines, cytokines, and growth
factors. Many cytokines are involved throughout a wound-healing and fibrotic response, with specific groups of genes activated
in various conditions. For example, chronic allergic airway disease in asthmatics is commonly associated with elevated type-2
cytokine profiles (IL-4, IL-5, IL-13, IL-9, IL-346) whereas IPF
patients more frequently present pro-inflammatory cytokine
profiles (IL-1, IL-1, TNF, TGF, and platelet-derived growth
factors (PDGF)47). Among many cytokines in various pulmonary fibrotic conditions, IL-4, IL-13, and TGF- have received
significant attention. Each of these cytokines can exhibit significant pro-fibrotic activity,48–51 acting through the recruitment,
activation and proliferation of fibroblasts, macrophages, and
myofibroblasts.2
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Type-2 inflammatory responses: pro-fibrotic IL-4 and IL-13.
IL-4, the archetypal type-2 cytokine, has been firmly established
as a pro-fibrotic cytokine and is elevated in IPF,52 cryptogenic
fibrosing alveolitis,53 radiation-induced pneumonitis and pulmonary fibrosis54 as well as liver fibrosis following infection
with Schistomsoma mansoni.55 IL-4 receptors are present on
lung fibroblasts49 with IL-4 signaling increasing extra cellular
matrix proteins and collagen deposition. Surprisingly, some
studies have suggested that IL-4 is superior to TGF-1 at inducing collagen synthesis from fibroblasts.49 Indirect mechanisms
of IL-4 include its ability to promote the alternative activation of
macrophages (AA-Mac), identified by the expression of arginase,56 Fizz-1,57 Ym-1,58 and mannose receptors.59 Macrophages
in general have long been associated with pulmonary fibrosis.
However, the precise mechanisms and functions of AA-Macs
in pulmonary fibrosis are only now being dissected. AA-Macs
can produce TGF-, PDGF60 and, through arginase upregulation, modulate polyamine and proline biosynthesis, cell growth,
and collagen formation.61 AA-Macs have been isolated and cultured from the bronchoalveolar lavage (BAL) of IPF patients,62
with culture supernatants from these AA-Macs significantly
increasing collagen production by normal human fibroblasts
in a CCL18-dependent manner. Animal studies have also identified the involvement of AA-Macs in several models of fibrosis,
including mice overexpressing human TGF in the lung,63 in
human and animal studies of dystrophic muscle fibrosis64 and
in multiple organ fibrosis following infection of IFNR − / − mice
with herpes virus.65 Although not identified as AA-Macs, macrophages in general have long been appreciated in human66 and
animal models of pulmonary fibrosis.67–69 Together these data
suggested that direct secretion of TGF, PDGF and proline by
AA-Macs are just a few of the many ways in which AA-Macs
influence the progression of pulmonary fibrosis.
Finally, one of the most renowned properties of IL-4 is its ability to promote the differentiation of T cells into Th2 cells, providing a source of many type-2 cytokines in this inflammatory
axis (IL-5, IL-9, IL-13, and IL-21). The Th2 cytokines interact
in dramatic ways propagating wound healing and potentially
pro-fibrotic responses. For example, IL-5 mobilizes, matures,
and recruits eosinophils,70 with IL-4 promoting TGF- production from eosinophils.37 IL-5 can also augment IL-13 production
and increase IL-13-dependent fibrosis.71 IL-9 can selectively
recruit and activate mast cells,72 with mast-cell-derived chymase increasing TGF activity and contributing to pulmonary
fibrosis.73 Mast cells can also promote fibroblast proliferation,
collagen, and MMP production,74 and may be involved in subepithelial fibrosis following allergen challenge.75 IL-21 can also
amplify Th2 pulmonary responses and IL-13-associated fibrosis
by upregulating IL-4/IL-13 receptor expression. Mice deficient
in the IL-21R showed reduced IL-13-dependent fibrosis following S. mansoni infection76 and reduced IL-13-mediated AHR in
a murine model of asthma, suggesting it may be an important
regulator of Th2-driven remodeling in the lung.77
IL-13 shares many properties with IL-4, due to common
receptor subunits (IL-4R), signal transduction pathways and
transcription factors (STAT-6). However, recent animal studies
106
have identified IL-4R-78 and STAT-679-independent IL-13associated responses, which may involve IL-13 signaling through
IL-13R2.80–82 Despite the common properties between IL-4
and IL-13, IL-13 has been identified as a key fibrogenic cytokine
in many fibrotic conditions (83, reviewed in 51) and can function
independently of TGF-.84 IL-13 can trigger the differentiation
of fibroblasts into -smooth muscle actin (-SMA) expressing
myofibroblasts and PDGF-producing cells85 with significant
mitogenic properties. Interestingly, IL-13-mediated differentiation of fibroblasts into myofibroblasts is refractory to steroid
inhibition, which may explain why steroids are not effective at
inhibiting fibrosis.
Pro-fibrotic IL-13 has been widely studied in animal models,
where gain of function experiments using a novel transgenic
approach (overexpressing IL-13), led to subepithelial fibrosis
accompanied by eosinophilic inflammation and mucus production,86 comparable to allergen-induced airway responses.
Similarly, loss of function studies, blocking or germ line deletion
of IL-13 but not IL-4, reduced collagen deposition following
exposure to aspergillus,87 OVA,88 bleomycin89,90 and FITC.91
It is important to note that the pro-fibrotic properties of IL-13
are not restricted to the lung, because hepatic fibrosis, following infection with Schistomsoma mansoni is also significantly
decreased following IL-13 blockade.91
In vitro culture of normal human fibroblasts with normal
human epithelial cells, which were pre-treated with IL-13,
produced significantly more TGF-, soluble and fibrillar collagen,92 supporting the notion that IL-13 can both directly and
indirectly promote collagen production by fibroblasts. Indeed,
fibroblasts isolated from IPF patients93 and allergic asthmatics94 demonstrate a hyper-responsiveness to IL-13, as well as
TGF and CCL-2, with significant interplay between these three
mediators.93 Several animal studies also propose a model where
IL-13, through various receptor subunits80,81 can induce plasminogen activator and MMP-9, enhancing the release of active
TGF95 and subsequent fibrosis. Together these human and animal studies indicate a coordinated and potentially combined
effect of IL-13 and TGF on fibroblast activation and collagen
deposition.40
Involvement of TGF in pulmonary fibrosis. TGF is derived
from most cell lineages derived from the bone marrow96 including T cells, macrophages,97 eosinophils, and neutrophils98 and
is one of the most widely studied pro-fibrotic cytokines. The
potent activity of TGF is regulated at the post-transcriptional
level by a latency-associated protein (LAP), which keeps TGF
in an inactive state. Dissociation of TGF from LAP is achieved
by many agents commonly found in fibrotic conditions, including cathepsins, plasmin,99 calpain,100 thromombospondin,96
integrin v6,101 and MMPs.102 These agents trigger the release
of biologically active TGF. Once active, TGF is incredibly
pleiotropic with growth and chemotactic properties, stimulating fibroblast proliferation and the synthesis of extracellular matrix proteins,50 recruiting inflammatory cells through
MCP-1 (CCL2)103 and suppressing T-cell responses. The various
and often opposing functions of TGF are likely explained by
its various sources and cellular targets.104 The inhibitory and
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suppressive properties of TGF were reviewed elsewhere,105–107
whereas this review focuses on the pro-fibrotic properties of
TGF.
Similar to the approach employed to study IL-13-mediated
fibrosis in the lung,86 active TGF has also been overexpressed
in the lungs of mice, with the development of severe interstitial
and pleural fibrosis, consisting of excess collagen deposition,
extracellular matrix proteins, fibronectin, elastin, and the presence of myofibroblasts.108 Interestingly, unlike IL-13 overexpression, TGF did not recruit inflammatory cells or enhance mucus
secretion in the lung, suggesting that TGF can directly induce
fibrosis in the absence of significant inflammation. Inhibiting
TGF activity, by interfering with SMAD-mediated signaling,109
significantly reduced dermal,110,111 renal,112–114 ocular,115
and pulmonary fibrosis.116,117 As mentioned above, TGFindependent84,118,119 as well as TGF- and IL-13-combined
mechanisms can contribute to wound healing and fibrosis.
Knowledge of the precise interactions and non-redundant compensatory pathways in addition to disease-specific dominance
of IL-13 and/or TGF could significantly improve therapeutic
options.
Chemokine cocktails in the fibrotic lung. Cytokine-producing
cells are efficiently recruited to sites of injury through chemokine gradients. Many chemokine gradients develop during
wound-healing responses, each recruiting specific chemokine
receptor-bearing cells; however, the CC and CXC chemokine families have received considerable attention in fibrotic
responses. For example, eosinophils bearing CCR3 and following CCL11 (Eotaxin) gradients and neutrophils, macrophages
and monocytes, bearing CCR2 and following IL-8 (KC in mice),
IL-17, CCL2 (MCP-1) and CCL3 (MIP1) gradients120 have
all been implicated in pulmonary fibrosis. CCL2, CCL3, and
CCL11 are themselves upregulated in pulmonary fibrotic conditions,121–127 with gene-deficient animal models confirming their
importance.124,128,129 However, a previously underappreciated
circulating cell, the fibrocyte, expressing CCR2,130 CCR3,131
CCR5,132 and CCR7,131 as well as CXCR4,133 represents a significant population of collagen-producing cells.134 The discovery of a rapid, ready, and plentiful supply of collagen-producing
fibrocytes from the bone marrow adds a new dimension to
pulmonary wound repair and fibrosis.135,136 Currently, there
are three potential origins of -SMA + myofibroblasts in lung
fibrosis; (1) resident interstitial fibroblasts differentiating into
collagen-secreting and extracellular matrix producing cells; (2)
a process of epithelial to mesenchymal transformation (EMT)
where local epithelial cells adopt fibroblast-like properties
and (3) the extravasation of circulating fibrocytes, originating
from the bone marrow and differentiating in the tissue into
myofibroblasts.137
During chronic injury, endothelial cells enter a process of
vasculogenesis (de-novo blood vessel formation) and angiogenesis (budding of new capillary branches from existing blood
vessels),138 laying down dense vascular beds permeating fibrotic
and regenerative tissue. Angiogenesis can be controlled by
several angiogenic factors including vascular endothelial
growth factor (VEGF), fibroblast growth factor, TGF, PDGF,
MucosalImmunology | VOLUME 2 NUMBER 2 | MARCH 2009
angiopoietin 1 (Ang1) and a vast array of cytokines139 and
chemokines.137
In particular, CXC chemokines identified as angiogenic or
angiostatic by their amino terminus, 3-aa sequence (Glu-LeuArg), known as the ELR motif, regulate the degree of neo-vascularization and remodeling. In general ELR + CXC chemokines
(CXCL1, 2, 3, 5, and 8), which bind to CXCR2, are angiogenic
and ELR − CXC chemokines (CXCL4, 9, 10, and 11), which
bind to CXCR3, are angiostatic. BAL fluid from IPF patients
is rich in ELR + CXC chemokines with a relative downregulation of ELR − CXC chemokines.139–141 Imbalanced ELR + and
ELR − CXC chemokine levels have also been observed in animal
models of pulmonary fibrosis,142–144 confirming observations
made in patients.
In summary, inflammation and the recruitment of circulating granulocytes, lymphocytes, monocytes, macrophages, and
fibrocytes, presents a continuous supply of pro- and anti-fibrotic
players, vital for efficient wound repair but potentially deadly
when not adequately controlled. Every step of this pathway
requires negative feedback loops that evoke significant control
over the entire process. An imbalance in chemokine production coupled with dysregulated cellular recruitment can result
in an excess of pro-fibrotic IL-13 or TGF, a surplus of myofibroblasts, and can convert a normal wound-healing response into
a pathological fibrotic reaction.
Phase III: tissue repair and contraction
The closing phase of wound healing consists of an orchestrated
cellular re-organization guided by a fibrin-rich scaffold formation, wound contraction, closure and re-epithelialization. The
vast majority of studies elucidating the processes involved in this
phase of wound repair have come from dermal wound studies
and in vitro systems. For this reason, we will extrapolate these
studies to the lung, where possible.
Myofibroblast-derived collagens and -SMA form the provisional extracellular matrix, with macrophage, platelet, and
fibroblast-derived fibronectin145,146 forming a fibrin scaffold.
Collectively, these structures are commonly referred to as granulation tissues. Primary fibroblasts or alveolar macrophages147
isolated from IPF patients produce significantly more fibronectin and -SMA than control fibroblasts,148 indicative of a state
of heightened fibroblast activation. Interestingly, IPF patients
undergoing steroid treatment had similar elevated levels of macrophage-derived fibronectin as IPF patients without treatment.
Thus, similar to steroid resistant IL-13-mediated myofibroblast
differentiation,85 macrophage-derived fibronectin release147 also
appears to be resistant to steroid treatment, providing another
reason why steroid treatment may be ineffective. From animal
models, fibronectin149,150 appears to be required for the development of pulmonary fibrosis, as mice with a specific deletion
of extra type III domain of fibronectin (EDA) developed significantly less fibrosis following bleomycin administration148
compared with their wild-type counterparts.
In addition to fibronectin, the provisional extracellular matrix
consists of glycoproteins (such as PDGF151), glycosaminoglycans
(such as Hyaluronic acid152), proteoglycans,153 and elastin.154,155
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Growth factor and TGF-activated fibroblasts migrate along the
extracellular matrix network and repair the wound. Within skin
wounds, TGF also induces a contractile response, regulating
the orientation of collagen fibers.156 Fibroblast to myofibroblast
differentiation, as discussed above, also creates stress fibers and
the neo-expression of -SMA,157 both of which confer the high
contractile activity158 within myofibroblasts. The attachment
of myofibroblasts to the extracellular matrix at specialized sites
called the “fibronexus” or “super mature focal adhesions” pull
the wound together, reducing the size of the lesion during the
contraction phase.159 The degree of extracellular matrix laid
down and, the quantity of activated myofibroblasts160 determines the amount of collagen deposition. To this end, the balance of MMPs to TIMPs161–163 and collagens to collagenases
vary throughout the response, shifting from pro-synthesis and
increased collagen deposition, towards a controlled balance,
with no net increase in collagen. For successful wound healing,
this balance often occurs when fibroblasts undergo apoptosis,
inflammation begins to subside, and granulation tissue recedes,
leaving a collagen-rich lesion. The removal of inflammatory cells
and especially -SMA + myofibroblasts is essential to terminate
collagen deposition.164 Interestingly, in IPF patients, the removal
of fibroblasts can be delayed, with cells resistant to apoptotic
signals,165–167 despite the observation of elevated levels of the
apoptosis inductor9 and FAS-signaling molecules.164 This relative resistance to apoptosis may potentially underlie this fibrotic
disease.160,168 However, it is important to note that several studies have also observed increased rates of collagen-secreting
fibroblast and epithelial cell169 apoptosis in IPF,170 suggesting
that yet another balance requires monitoring—that of fibroblast
apoptosis and fibroblast proliferation. The signals which initiate
fibroblast apoptosis in IPF, are not very well understood with
several factors postulated, such as cytokine imbalances, genetic
causes, and constitutive anti-apoptotic pathways160,165,170,171
similar to some cancerous cells.
From skin studies, re-epithelialization of the wound site reestablishes barrier function and allows encapsulated cellular
re-organization. Several in vitro and in vivo172 models, using
human173 or rat174 epithelial cells grown over a collagen matrix,
or tracheal wounds in vivo, have identified significant stages of
cell migration, proliferation,175 and cell spreading. Rapid and
dynamic motility and proliferation, with epithelial restitution
from the edges of the denuded area172,173,176 occur within hours
of the initial wound. In addition, sliding sheets of epithelial cells
can migrate over the injured area177,178 assisting wound coverage. Several factors can regulate re-epithelialization with serumderived TGF174 or MMP-7179,180 (which itself is regulated by
TIMP-1181) appearing to play significant roles.
Collectively, the degree of inflammation, angiogenesis, and
amount of extracellular matrix deposition all contribute to the
net collagen deposition and ultimately whether a fibrotic lesion
develops. Therapeutic intervention, interfering with fibroblast
activation, proliferation or apoptosis requires a thorough understanding and appreciation of all of the phases of wound repair.
Although these three phases are often presented sequentially,
during chronic or repeated injury these processes function in
108
parallel, placing significant demands on regulatory mechanisms.
ETIOLOGY AND PATHOGENESIS OF COMMON PULMONARY
FIBROTIC DISEASES
Alleviating symptoms is the primary concern of patients presenting pulmonary fibrosis. Understanding the etiology of pulmonary fibrosis can provide long-term symptomatic relief and
possible reversal of the disease. To this end, there are currently
several well-known risk factors associated with pulmonary fibrosis that will be described below. In many cases, animal models
have obvious advantages in studying the regulatory mechanisms
in pulmonary fibrosis and airway remodeling.
Cystic fibrosis and cystic lung disease
With regard to etiology, cystic fibrosis (CF) is unique among
pulmonary fibrotic conditions and can be attributed to a single
gene mutation making it the most common monogenic disease
of Caucasians, affecting 1 in 2,500–4,000.182
CF transmembrane conductance regulator (CFTR),183 is the
genetic “Achilles heal” responsible for the disease. The CFTR
protein product is a chloride channel protein found in the
membrane of cells lining the lungs, as well as the liver, pancreas,
intestines, reproductive tract, and skin.184–186 However, the leading cause of mortality in humans with CF is lung disease.184
In addition to direct effects of CFTR mutations, resulting in
deficient cAMP-mediated chloride secretion across epithelia
and dysfunctional mucus regulation, CF patients are prone to
progressive pulmonary damage, submucosal inflammation, and
increased susceptibility to bacterial infection.187 Long-term
aerosolized antibiotics may limit bacterial colonization;188–190
however, a consequence of chronic infection is recurring lung
injury, chronic inflammation,191,192 airway remodeling,193 and
fibrosis. The chronic inflammatory response, in particular the
neutrophilic response, is a significant feature driving pathology in CF. Gaggar et al.194 recently identified that neutrophil
elastase, an enzyme that is significantly elevated in BAL fluid
from CF patients, can promote pro-MMP9 and inhibit TIMP1, thereby disrupting the protease/anti-protease balance.194–196
In addition, epithelial cell regeneration and repair may also
be disrupted, accounting for altered lung physiology in CF.197
Several cftr − / − mice have been developed with varying degrees
of lung disease, identifying CF-modifying genes within different founding lines.198–200 The monumental task of developing a
mouse that spontaneously develops lung disease was achieved,
allowing the pathophysiological dissection of murine CF. cftr − / −
mice develop parenchymal interstitial thickening and fibrosis
with granulocyte influx, fibroblast infiltration and the deposition
of matrix proteins.199 The development of a mouse model of CF
has allowed interesting studies addressing anti-inflammatory
responses,201 the involvement of modifier genes,202,203 the impact
of bacterial infection204,205 and multi-organ complications.206
Radiation and chemotherapy-induced lung injury
Thoracic radiation therapy (RT) is used to treat lung, eosophageal, breast, and lymphoid cancers. However, a common
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dose-limiting complication of RT is the development of pulmonary interstitial injury and inflammation, often referred to
as radiation pneumonitis and emergence of fibrotic foci.207–209
Multiple mechanisms have been identified in RT-induced
fibrosis, including alveolar damage,210 increased reactive oxygen species (ROS) and the toxic effects of ROS on parenchymal
cells,211,212 disruption of proliferation-associated transcription
factors,213 and the influx of inflammatory cells, such as macrophages and lymphocytes.214,215 Furthermore, dysregulated
pro-inflammatory and pro-fibrotic cytokines, TGF, IL-6,
MMPs,216–220 and chemokines,221 in addition to reduced antiinflammatory cytokines following radiation222 can further exacerbate the inflammatory and wound-healing response. Animal
models have revealed genetic determinants of RT-induced fibrosis213,223 corresponding with similar genotype-related associations in humans.224 Collectively, RT of the thoracic region can
cause significant damage to radiation-sensitive alveolar regions
of the lung invoking a dysregulated inflammatory cascade, rich
in pro-inflammatory and pro-fibrotic mediators. Dysregulated
chemokines, transcription factors, and anti-inflammatory
pathways can further compound this uncontrolled response,
leading to pulmonary fibrosis.
Similar to radiation therapy, chemotherapy can cause lung
injury with variable consequences depending on dose rate,
duration, pre-existing lung disease, and concomitant steroid
use.225,226 The Streptomyces verticullatus-derived antibiotic,
bleomycin (BLM),227 is effective against squamous cell carcinomas and skin tumors;228 however, like RT, an unfavorable side
effect involves inflammatory and fibrotic responses in the lung.
BLM-induced inflammation occurs in up to 46% of patients
treated229 with complications in the lung and skin due to a lack
of the endogenous bleomycin-inactivating enzyme, bleomycin
hydrolase, in these tissues.230
Our understanding of BLM-induced fibrosis has been assisted
by the development of animal models, which reproduce many,
but not all, of the characteristics of the human disease.230 BLM
can directly cause cell death231 and reduce O2 into free radicals, causing DNA breakage.232 Depending upon the route of
administration, epithelial and endothelial cells are some of the
earliest cells affected,233 causing a leukocyte-rich inflammatory
response. Blockade of this inflammatory response in animal
models, with anti-CD11 Ab-inhibiting cellular extravasation,
dramatically reduced pulmonary collagen and fibrosis, demonstrating the significant contribution of inflammatory cells on
the resulting fibrotic response.234 The inflammatory cytokines,
TNF,235 IL-1,236 IL-6237 and pro-fibrotic TGF238,239 are
accompanied by FAS-L-expressing cells, leading to more apoptosis.16,240 Blockade of TNF, IL-1, FAS-Ligand or TGF can
reduce the inflammatory and resultant fibrotic response following BLM administration.235,240–242 Thus, TNF, IL-1, IL-6,
and TGF are some of the possibly many mediators involved in
BLM-induced fibrosis. The BLM model has been used to dissect
the involvement of many cytokines in the pulmonary fibrotic
response. The involvement of type-2 cytokines is less clear, with
IL-4 and IL-5 playing no significant role,243–245 whereas IL-13,
either directly90 or indirectly through TGF,80,81 contributes
MucosalImmunology | VOLUME 2 NUMBER 2 | MARCH 2009
to the fibrotic response. There is also evidence that Type-1
cytokines are involved,246 with fewer inflammatory cells, lung
hydroxyproline content, weight loss, and mortality observed in
IFN − / − mice.247 Blocking the IFN-promoting cytokine IL-12
or germ line deletion of IL-12248 yielded similar results.248 BLM,
although invoking a significant inflammatory response, can also
promote fibroblast proliferation249 and TGF production from
endothelial cells250 directly. Thus, BLM appears to have multiple
properties, directly causing cell death and apoptosis, invoking an
inflammatory response and promoting fibroblast proliferation
and TGF production. For these reasons, the mouse model of
BLM-induced fibrosis provides a great tool to dissect the relative contribution of the many pathways, cells, and mediators
involved in drug-induced fibrosis.
Asthma and allergic airway inflammation
The number of individuals suffering from allergic airway
inflammation and asthma has seen an unprecedented growth
over the past 30 years, particularly within the urban areas of
both developed and developing countries.251 Allergic asthma
is a polygenic disease,252 characterized by allergen-specific IgE
and IgG1, airway and interstitial eosinophilia, mucus secretion
and airway hyper-reactivity.253 Chronic asthma with repeated
bouts of allergen exposure and dysregulated inflammation at
mucosal surfaces can lead to goblet cell hyperplasia, smooth
muscle hypertrophy and hyperplasia, angiogenesis and ultimately subepithelial fibrosis.254–257
CD4 + Th2 cells orchestrate many aspects of the allergic
inflammation, driven by dendritic cell or basophil-derived
IL-4 and IL-25.258–263 Activation and egression of cytokinesecreting Th2 cells into the interstitium and mucosal surfaces
of the lung propagate local cellular influx. More specifically,
Th2-derived cytokines, IL-5 and IL-9, mobilize, mature and
recruit eosinophils and mast cells70,264,265 into the tissue and
airspaces, and these cells are typically found in biopsies of asthmatic individuals. TGF is also significantly elevated in human
asthmatics41,266–270 with the degree of subepithelial fibrosis correlating with a loss of forced expiratory volume (FEV1). These
observations of increased eosinophils, TGF and subepithelial
fibrosis led Flood-page et al.271 to study the specific cellular
source of TGF. Indeed, 86% of TGF mRNA + cells in the
bronchial mucosa of asthmatics were eosinophils, distinguishing eosinophils as a significant source of pro-fibrotic TGF in
the allergic lung.39 Furthermore, several studies have identified
correlated collagen deposition with increased numbers of tissue
eosinophils and myofibroblasts19,38 as well as the expression of
submucosal MMP9 and MMP12.272
These observations have led to several clinical trials and
treatment regimens using anti-IL-5 antibodies to block tissue
eosinophilia with few successes. Treatment of allergic asthmatic
patients, as well as atopic dermatitis patients,273 with anti-IL-5
antibodies (mepolizumab) led to significant reductions in tissue
eosinophilia,271,274 despite no change in late-phase cutaneous
allergic reactions. Most striking was a reduced thickness and
density of the extracellular matrix (tenascin, lumican, and procollagen III (COL3A)) following anti-IL-5 treatment, suggesting
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that IL-5-mediated tissue eosinophilia was indeed responsible
for ECM deposition. However, despite these encouraging results,
the precise role and involvement of eosinophils in human asthma
is debated, with many clinical trials of anti-IL-5 mAb reporting
little to no clinical improvement.275,276
Animal studies, using either IL-5-deficient mice277 or eosinophil-ablated mice278,279 have supported a significant role for
eosinophils, with reduced airway remodeling, including peribronchial fibrosis and smooth muscle thickness, in addition to
several other features of allergic asthma following chronic airway
exposure. Similarly, blocking TGF280 or interfering with TGF
signaling281 could also significantly attenuate airway remodeling
following chronic allergen exposure.
Taken together, animal models have demonstrated a clear role
for eosinophils and eosinophil-derived TGF in airway damage and remodeling. Human studies, however, have produced
a spectrum of results and require additional studies, with welldefined end points to address the role of IL-5 and eosinophils
in the progression and resolution of subepithelial fibrosis in
asthmatic airways.
IL-13 may also be a damaging cytokine in allergic individuals.
Many of the pathological conditions identified in allergic asthmatics can be traced to IL-13. For example, IL-13 can mediate
goblet cell hyperplasia in local epithelia282 and increase mucus
production92 that can block the small airways.283,284 IL-13 can
also promote epithelial repair,285,286 fibroblast growth,85,287
EMT,288 and collagen deposition.92 Beyond the airway epithelium, IL-13 also causes smooth muscle hyperplasia289 and subepithelial fibrosis.88 Similar to mechanisms proposed using the
bleomycin model, IL-13 can synergize with and promote profibrotic TGF290,291 eotaxin production,40 and TIMP expression.292 Thus, within the context of allergic asthma, eosinophils,
TGF, and IL-13 may all contribute to airway remodeling and
pulmonary fibrosis.
Less common pulmonary fibrotic conditions with known
etiologies
Environmental particulates from smoking or occupational
exposure can have toxic effects on the mucosal surfaces of the
lung. For example, jobs that involve mining or that expose workers to asbestos, metal dusts, or silica dust can cause pulmonary
fibrosis.293 Agricultural workers can also be affected,293 with
exposure to organic and inorganic substances,294,295 fumes,296 or
moldy hay297 causing allergic inflammation and fibrosis, often
referred to as Farmer’s Lung.298–300 Granulomatous lung disease and sarcoidosis is less common, with a global incidence of
16.5–19/100,000.301 These diseases are significantly influenced
by genetic and environmental factors. To date, the causative
agents have not been identified.302 An alveolar macrophage
gene-transcript profile303 that is similar to Mycobacterium
tuberculosis infection has led to the hypothesis that bacteria
may be involved. However, to date, bacteria have not been isolated from sarcoidosis patients. Chronic inflammation and the
development of inflammatory cell-rich pulmonary granulomas,304,305 rich in type-1 cytokines and chemokines122,306–309
and T cells310 can dramatically disrupt parenchymal architec110
ture, endothelial cells and the alveolar spaces of sarcoidosis
patients. Immunohistochemical analysis of human and animal
lung biopsies and post-mortem histological sections have identified elevated collagen and fibronectin in granulomas of sarcoidosis patients.305 Furthermore, co-expression of pro-fibrotic
TGF within the granulomas was also observed in sarcoidosis
granulomas.311–313 Despite these varying etiologies, recurring
lung injury and inflammation314 is common to many of these
fibrotic conditions, and may broadly underlie the pathogenesis
of pulmonary fibrosis.
Idiopathic pulmonary fibrosis
When all known causes of interstitial lung disease and fibrosis have been ruled out, the condition is referred to as “idiopathic” (of unknown origin) pulmonary fibrosis (IPF). Despite
an unknown etiology, there are a number of conditions and
risk factors associated with the disease including; smoking,315
farming, and occupational hazards316,317 and viral, and bacterial infections.318–320 Furthermore, in one study, IPF patients
had a greater propensity to develop primary lung cancer,
compared with non-IPF patients with chronic lung disease or
patients without lung disease.321 Reports of familial aggregation
of IPF also suggest that there may be a genetic component
to IPF.322,323 As mentioned above, the incidence of IPF in
the United States is 30/100 0004 and 34 000 new cases annually,5 with a similar increasing incidence of IPF in the United
Kingdom.324
IPF is characterized by usual interstitial pneumonitis325 and
progressive interstitial fibrosis caused by excessive extracellular matrix deposition. Regions of fibroblast and myofibroblast
accumulation, specifically between the vascular endothelium
and alveolar epithelium disrupt the architecture of the lung,
giving a “honeycomb” appearance.321 The pathogenesis of IPF
has been debated for many years with two different schools
of thought. One group suggests an inflammatory stimulus is
involved, with recurring inflammation leading to immunopathology, tissue destruction and the propagation of a woundhealing response.32,36,326,327 Others suggest a slightly different
pathogenic mechanism in which an initial or absent inflammatory stage is quickly followed by an uncontrolled wound-healing
response.30,328 Central to the argument negating the dominant
role of inflammation is the inefficiency of corticosteroids and
other anti-inflammatory agents to control IPF34,35 despite
some reports of enhanced survival.329 Furthermore, the ability of epithelial cell-derived TGF330,331 to invoke a fibrotic
cascade with increased interstitial collagen and fibroblast
proliferation in the absence of inflammation further support
these views. We believe the controversy reflects our limited
knowledge and insight into the causative agent(s) and pathogenesis of IPF. A common, and accepted view is the early role
of inflammatory events, initiating a wound-healing response.
Whether the dysregulated wound-healing response continues
in the absence of subsequent inflammation or not has yet to be
clarified. Continuous chemokine and cytokine production47 in
diagnosed IPF patients indicates that damage and subsequent
inflammation may be ongoing.
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The cytokine profile from biopsy or BAL-derived cells
or BAL fluid of IPF patients is rich in pro-inflammatory
cytokines; IL-1 ,332,333 IL-8,122 IL-18,334 TNF ,335 MCP1122,336as well as Type-2 cytokines, and their receptors.337,338 The
mixed cytokine profile, derived primarily from inflammatory
cells67 and leukocytes,33,339 can have significant effects on all
aspects of wound healing including vascular remodeling, myofibroblast differentiation, EMT, TGF, and IL-13 production. In
addition to the direct fibroblast-activating properties of TGF
and IL-13, co-expression of these two cytokines in IPF has been
observed.93 Fibroblast hyperplasia170 and the reduced expression of apoptotic mechanisms (bcl-2 and membrane FAS-L)340
in IPF can further augment the fibrotic response. Collectively,
a cascade of failed regulatory mechanisms and hyper-secretion
of cytokines, chemokines and growth factors,47 culminates in
an out-of-control fibroblast-mediated wound-healing response.
Physiologically, IPF can dramatically compromise oxygen diffusion, lung function341 and is typically a fatal disease.
REGULATION OF PULMONARY FIBROSIS
It is becoming clear that an imbalance of stimulatory cytokines,
chemokines, and growth factors likely over-activate resident
parenchymal and circulating cells and may underlie the over
exuberant wound-healing responses that lead to fibrosis. In a
normally controlled cellular response, negative feed back loops,
anti-inflammatory molecules, inhibitory receptors, and apoptotic pathways operate to fine tune and terminate responses once
a desired outcome is achieved. Common to many pulmonary
fibrotic conditions with both known and unknown etiologies
may be a break down in these regulatory mechanisms, resulting in an excessive inflammatory cascade, neo-vascularization,
uncontrolled fibroblast activation, and fibrosis. In this section
of the review we will highlight some of these endogenous regulatory mechanisms that either operate endogenously or can be
exploited therapeutically to counter balance the uncontrolled
responses.
Regulation of inflammatory responses: Tregs and IL-10
T cells with the primary function of attenuating immune
cell activation and proliferation are frequently referred to as
regulatory T cells (Treg). Although the specific details of antigen
specificity, precise mechanisms of suppression, and distinguishing features continue to grow, their role in fibrotic responses
have been under studied. However, given their ability to dampen
inflammatory responses, the ability of Tregs to interfere with
upstream events and slow the progression of fibrosis has been
implied. In particular Treg-derived IL-10, and other surface
molecules342 although not exclusively derived from Tregs, can
function as a general immunosuppressant343 and control fibrosis.342 Polymorphisms in the signal sequence of the IL-10 gene
have been identified in IPF patients, corresponding to reduced
IL-10 production, suggesting that endogenous anti-inflammatory mechanisms may be impaired in this condition. Supporting
this notion, loss of function studies using LPS-induced lung
injury and fibrosis in IL-10-deficient mice led to significantly
stronger inflammatory responses with greater subepithelial
MucosalImmunology | VOLUME 2 NUMBER 2 | MARCH 2009
thickening and extracelular matrix protein content.344 In gain
of function studies, induction of IL-10 significantly reduced
collagen deposition following bleomycin administration in
murine models.345 Following IL-10 gene delivery BAL fluid
TNF and neutrophil-derived MPO levels were significantly
reduced, with another similar study observing reduced macrophage-derived TGF,43 suggesting that IL-10 inhibits inflammatory cell recruitment.345 Corroborating these findings, Dosanjh
et al.346 reported higher levels of IL-8, with reduced IL-10 levels in the BAL fluid of cystic fibrosis patients. Thus, IL-10 can
attenuate the inflammatory events upstream of the fibrotic pathway. In addition to suppressing inflammatory events, IL-10 can
act directly on fibroblasts, reducing TGF-induced collagen
production.345 Following lung injury in a rat model of radiation-induced fibrosis, pneumocytes in the epithelial layer of
the lung had reduced expression of IL-10, compared to control
lungs, which may permit greater local inflammation.222 Thus,
IL-10 immunotherapy, with a sound understanding of timing
and when to dampen inflammatory events may hold promise
for pulmonary fibrotic conditions.347 Beyond the lung, IL-10
has been shown to regulate kidney348 and liver349 inflammation, and fibrosis.350 Therapeutically manipulating IL-10,
in particular endogenous IL-10-producing cells which may
be present but in too low frequencies to significantly halt the
inflammatory onslaught, may be a useful avenue to pursue. This
has been demonstrated successfully in models of allergic airway
inflammation, where a reduction in airway and tissue inflammation, mucus production, and airway hyper-responsiveness
was observed.351
IL-13R2 and LAP: endogenous attenuators of fibrosis
As dis c uss ed throughout this re vie w, TGF and
IL-1351,84,85,352,353 are dominant pro-fibrotic cytokines, activating fibroblasts, and promoting differentiation into -SMAproducing myofibroblasts and collagen production. Thus, tight
regulation and fine-tuning of these two potent molecules is
essential. Two molecules that can serve this very purpose are
the IL-13R2, an endogenous decoy receptor that attenuates
IL-13 activity and, as discussed earlier, LAP, a latency-associated protein, which keeps TGF in an inactive state. To our
knowledge there are no studies to date reporting the specific
induction of endogenous LAP to attenuate TGF bioactivity;
however, introduction of exogenous recombinant LAP could
theoretically be used to attenuate TGF activity354 (similar to
anti-TGF antibody280). Upregulation of a TGF-binding protein, endoglin, however has been observed in animal models of
renal fibrosis.355 Exploiting this pathway to attenuate TGF may
be another option. Although the exact mechanism is unknown,
the introduction of taurine and niacin into the diet of small
rodents attenuates BLM-induced fibrosis, apparently by reducing TGF production,356 suggesting that dietary supplements
may be useful therapeutics. Disrupting TGF-associated ROS357
or other downstream TGF-signaling pathways358 also hold
promise.
IL-13R2 is expressed predominantly by non-hematopoietic
cells (unpublished observations) and attenuates IL-13 activity
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in vivo.359 Given that IL-13 can act at multiple stages of the
inflammatory and wound-healing response,51,92,284,288,358,360
it comes as little surprise that attenuation of IL-13 can have
profound effects on the degree of pulmonary inflammation
and fibrosis in many pulmonary disease models.88,90,361 Several
methods of IL-13 attenuation have been described, including
neutralizing Abs,87 treatment with sIL-13R2,359,362–364 or targeting the IL-13R2-expressing cells. The conclusions from all
of these studies indicate that targeting the IL-13 pathway holds
great promise for the treatment of fibrosis.
Resetting the imbalance
An imbalance of cytokines, chemokines or cells can disrupt
many downstream processes (Figure 2). For example, an imbalance between collagen-catabolizing MMPs and their specific
inhibitors, TIMPs, can result in excessive collagen breakdown.
However, they can also promote TGF activation in peripheral
cells.365–367 Increased TGF can further feed back to induce
more MMPs368 and promote EMT.368 Thus, a breakdown in
one process (MMP production) can quickly catalyze and
disrupt other regulatory mechanisms (TGF responses). Within
mammalian systems, a refined balance between “on” and “off ”
signals is critical to maintain homeostasis. In a dysregulated
wound-healing response several key mechanisms appear to be
off balance (Figure 2).
(1)
Inflammation “vs.” Immunosuppression. Excessive or
recurring inflammatory events can cause excessive wound-healing responses that lead to the development of fibrosis. Either
eliminating the causative agent, such as allergen avoidance, or
treatments with anti-inflammatory agents such as corticosteroids may help restore the balance.
(2)
MMP “vs.” TIMP. MMPs can disrupt the basement
membrane and allow the influx of inflammatory cells. Inhibiting
MMP activity could be detrimental in immunity and in the process of re-epithelialization;369 however, in pathological fibrotic
responses, neutralization of specific MMPs either with small
molecules,157 inhibitors157 or by influencing TIMP expression
may help restore this imbalance.
(3)
Fibroblast apoptosis “vs.” proliferation. The late-stage
apoptosis of fibroblasts is required for successful wound healing
ANTI-INFLAMMATORY
CYTOKINES
ANGIOSTATIC,
CXC ELR–CHEMOKINES
FIBROBLAST APOPTOSIS
TIMP’S
APOPTOSIS
PRO-INFLAMMATORY
CYTOKINES
INFLAMMATION
ANGIOGENIC, CXC ELR+ CHEMOKINES
-SMA+ MYOFIBROBLASTS
MMP’S
COLLAGEN DEPOSITION
Figure 2 Imbalanced wound-healing response. For successful wound healing, a regulated response is maintained through negative feedback loops
and a balance of catabolising and regenerative processes. Several imbalances may develop and lead a normal healing response into a fibrotic
cascade. Excessive inflammation and the production of inflammatory and fibroblast-activating cytokines, through a breakdown in anti-inflammatory
mechanisms can develop. Over-production of angiogenic CXC ELR + chemokines, the recruitment of fibrocytes and increased frequency of -SMA +
cells in the injury site can result in too much collagen deposition. Resetting the balance with targeted therapeutics (i.e., cytokine-blocking antibodies)
may help slow the progression of fibrosis.
112
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REVIEW
and termination of collagen deposition. As mentioned above,
resistance to apoptosis has been observed in fibroblasts from
IPF patients.165–167 Restoring fibroblast apoptotic pathways or
selectively depleting fibroblasts at the appropriate time may help
slow the progression of fibrosis. Modulating local cytokine and
growth factor levels could also influence fibroblast proliferation
and activation indirectly.
(4)
ELR + “vs.” ELR − CXC chemokines. The prolonged induc+
tion ELR chemokines, due to inflammatory signals can lead to
excessive vascularization. Anti-angiogenic therapy,370 an area
actively pursued in cancer therapy was recently investigated in
fibrotic conditions.371 Inhibiting VEGF or promoting endostatin and anastellin (endogenous inhibitors of angiogenesis) may
limit inflammation and the recruitment of myofibroblasts.
Neutralizing angiogenic ELR + CXC chemokines or enhancing angiostatic ELR − CXC chemokines,372–374 in combination
with other therapeutic interventions, may also dramatically
halt the inflammatory cascade and avoid the requirements for
angiogenesis.
CONCLUSION
Pulmonary wound repair is an extremely dynamic process
intersecting immunology, structural biology, and airway
physiology. For successful repair a collaborative effort between
these systems is essential. Dysregulation in one response
can have ripple effects on others and progressively turn a
well-choreographed healing response into a fibrotic lesion.
Vascular damage must be quickly repaired with a fibrin-rich
clot. This is followed by an influx of inflammatory cells. Chronic
or recurring inflammation requires rapid resolution to avert
immunopathology while providing the necessary cellular
participants. Parenchymal cells that are responsive to inflammatory cues must proliferate and migrate into the damaged
area, restore tissue architecture, with the inflammatory cells
ultimately undergoing apoptosis to prevent excessive collagen
deposition.
In pulmonary fibrotic disease states, the development
and progression of the healing response has slipped out of
control, disrupting many delicate balances. As discussed in this
review, there are a number of compensatory and redundant
processes, which all contribute to proficient healing and remodeling. With this in mind, and despite significant advances in
our understanding of these pathways and balances, there
is a lack of therapeutic intervention and new therapies for
pulmonary fibrosis. Further studies are required to elucidate
the roles of the many mediators (cytokines, chemokines and
growth factors) observed in both human and animal models
of pulmonary fibrosis. Greater still, pre-clinical and clinical
investigations with chemokine receptor antagonists, angiogenesis inhibitors and Abs to the pro-fibrotic molecules IL-13 and
TGF are required. A combined effort by clinicians and lab
researchers across platforms and disciplines could make this a
climbable mountain.
DISCLOSURE
The author declared no conflict of interest.
MucosalImmunology | VOLUME 2 NUMBER 2 | MARCH 2009
ACKNOWLEDGMENTS
This review was improved by peer-review and funded by the Intramural
Research Program at the NIH/NIAID. Owing to space and word
limitations, we apologize to the many researchers whose work we have
not mentioned in this review, but who have significantly contributed to
our current understanding of pulmonary fibrosis. We also thank Dr Allen
Cheever for helpful comments.
© 2009 Society for Mucosal Immunology
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